The long term objective of this research is to understand the biogenesis of peroxisomes in molecular detail. Peroxisomes are nearly ubiquitous in eukaryotic cells an have essential functions, including fatty acid catabolism an the first steps in plasmalogen biosynthesis. Peroxisomes grow by the posttranslational import of newly-synthesized proteins. Three types of targeting information may direct proteins to peroxisomes: noncleaved C-terminal tripeptides (typically SKL), N-terminal cleaved oligopeptides, or internal topogenic domains. Import involves receptors (one for SKL identified) and a translocation apparatus that requires ATP hydrolysis. New peroxisomes from by division of preexisting ones. Several fatal inherited disorders (Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease) are cause by defective peroxisome biogenesis; peoxisomal membranes assemble, but the import of matrix proteins does not occur. Peroxisomes in S. cerevisiae and are required for growth on, fatty acids. We developed a positive selection procedure with which we have identified novel mutants that are defective in peroxisome biogenesis (peb). Two apparently lack peroxisomes and resemble Zellweger syndrome. Two have clusters of normal-looking peroxisomes which are selectively defective in the import of subsets of peroxisomal proteins. The data imply the existence of three branches in the peroxisomal import pathway (or three pathways). We have cloned and sequenced a previously unknown gene that complements one of the new mutants. We have also purified peroxisomes of S. cerevisiae and established as in vitro import system with them. The specific aims, in brief, are; A. Clone and analyze the genes required for peroxisome biogenesis that are defective in our peb mutants. B. Determine the properties, intracellular locations and functions of the peroxisome biogenesis gene products. C. Analyze the cellular defects in the mutants. D. Investigate the topological and functional relationships between proteins that participate in peroxisome assembly. E. Identify and characterize the corresponding human genes, if possible. F. Isolate additional mutants with new properties. These studies should provide fundamental new information about an intriguing problem in cell biology, peroxisome biogenesis, for which limited molecular information is yet available. They may also shed light on several human diseases in which peroxisome assembly in defective.